PROJECT SUMMARY Infertility affects 10% to 15% of couples worldwide, and a male factor contributes to around 50% of these cases. Male infertility is diagnosed predominantly on the results of standard semen analysis, which provides information about sperm count, morphology, and motility. However, many sperm samples from infertile men pass this standard analysis but, for unknown reasons, still lack the ability to fertilize an egg. Thus, to improve male fertility, our long-term goal is to reveal the basic mechanisms by which sperm become competent to fertilize an egg and to identify new diagnostic strategies and therapeutic targets. In many mammalian species membrane hyperpolarization (when the intracellular voltage becomes more negative) is a key event in sperm becoming competent to fertilize an egg (capacitation). We previously showed that sperm from mice that lack the sperm-specific SLO3 K+ channels cannot undergo membrane hyperpolarization and are infertile. Hyperpolarization is also associated with human sperm capacitation, and a depolarized membrane is associated with impaired fertilization capacity in human sperm. However, the ion channels responsible for regulating membrane potential in human sperm are uncertain. In this proposal we aim to determine the ion permeabilities that underlie membrane potential changes in human sperm and how membrane hyperpolarization regulates changes in intracellular calcium (another key aspect of sperm capacitation). We propose that human sperm membrane potential is regulated by the potassium (K+) channel SLO3 as in mouse sperm and that SLO3 dysfunction might be responsible for some cases of male idiopathic infertility. These studies might also produce a valuable clinical tool ? measurement of sperm membrane potential using voltage sensitive dyes - to predict human sperm fertilization capacity. Additionally, if SLO3 plays an essential role in human fertilization in humans as it does in mice, this sperm-specific channel would provide a new, non-hormonal target for a male contraceptive.